Reticulocyte lysate assay for in vitro translation and posttranslational modification of Ras proteins.
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Alkyl and Aryl Transferases↗
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Posttranslational modifications of endothelial nitric oxide synthase.
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Acylation↗
Biosynthesis of lipoic acid and posttranslational modification with lipoic acid in Escherichia coli.
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Bacterial Proteins↗
Senile plaque composition and posttranslational modification of amyloid-beta peptide and associated proteins.
Amyloid deposits are primarily composed of the amyloid-beta protein, although other proteins (and metal ions) also have been colocalized to these lesions. The pattern of oxidative modifications in amyloid plaques is very different to that associated with neurofibrillary tangles and neuronal cell bodies, likely reflecting the different composition of these structures, accessibility of oxidants, the generation of oxidants in and around these structures and the intrinsic antioxidant defense systems to protect these structures. Future studies directed at understanding Abeta interactions with other amyloid components, the role of oxidative modifications in stabilizing amyloid deposits and the determination of protease cleavage sites on Abeta may provide mechanistic insights regarding both amyloid formation and removal.
Amyloid beta-Peptides↗
Mono-ADP-ribosylation: a reversible posttranslational modification of proteins.
Mono-ADP-ribosyltransferase activity has been detected in numerous vertebrate tissues and transferase cDNAs from a few species have recently been cloned. In vitro ADP-ribosylation has been demonstrated with diverse substrates such as phosphorylase kinase, actin, and Gs alpha resulting in the alteration of substrate function. ADP-ribosylation of endogenous target proteins has been observed in chicken heterophils, rat brain, and human platelets, and integrin alpha 7 was found to be the endogenous substrate of the GPI-anchored rabbit skeletal muscle transferase. The reversibility of ADP-ribosylation is made possible by ADP-ribosylarginine hydrolases which have been isolated and cloned from rodent and human tissues. The transferases and hydrolases could in principle form an intracellular ADP-ribosylation regulatory cycle. In the case of the skeletal muscle transferases, however, processing of ADP-ribosylated integrin alpha 7 is carried out by phosphodiesterases and possibly phosphatases (Fig. 1). Most bacterial toxin and eukaryotic mono-ADP-ribosyltransferases, and perhaps other NAD-utilizing enzymes such as the RT6 family of proteins, share a common catalytic-site structure despite a lack of overall sequence identity. The transferases that have been studied thus far possess a critical glutamic acid and other amino acids at the catalytic cleft which function to position NAD for nucleophilic attack at the N-glycosidic linkage for either ADP-ribose transfer or NAD hydrolysis. The amino acid differences among transferases at the active site may reflect different catalytic mechanisms of ADP-ribosylation or may be required for accommodating the different ADP-ribose acceptor molecules.
ADP Ribose Transferases↗
Acylation of myelin basic protein peptide 1-21 with alkyl carboxylates 2-10 carbons long affects secondary structure and posttranslational modification.
A peptide consisting of the first 21 residues of human myelin basic protein (MBP) was synthesized. The N-terminal alanine of portions was blocked in separate experiments with alkyl carboxylates varying in size from 2 to 10 carbon atoms. The effects of these different alkyl carboxylates at the N-terminus on the secondary structure was studied by circular dichroism (250-190 nm). In water, the spectra of the unblocked peptide suggested unordered structure with large negative ellipticities at 198 nm. Addition of an acetyl group altered the magnitude of [theta]198 from -21856 +/- 2319 to -11095 +/- 1000 deg cm2 dmol-1, suggesting a significant increase in ordered structure. When peptides with longer alkyl carboxylates, acylated at the N-termini, were studied, the magnitude of theta 198 approached that of the unblocked peptide but greater negative ellipticities were observed for the C8 and C10 alkyl carboxylates. The theta 222 values were generally low (-1803 +/- 463) but increased with increasing length of the alkyl carboxylate to about -3200 deg cm2 dmol-1, suggesting that little alpha-helical structure was present. The spectra were also taken in lipid-mimetic solvents, including 2-propanol, methanol, and lysophosphatidylglycerol (lysoPG). In general the theta 198 and theta 222 values were suggestive of increased structure in these environments compared to water. In 90% 2-propanol the theta 198 of the unblocked peptide did not change when an acetyl group was added to the N-terminus (9088 compared to 8477 deg cm2 dmol-1). Addition of longer alkyl carboxylates correlated with larger, negative ellipticities.(ABSTRACT TRUNCATED AT 250 WORDS)
Acylation↗
Posttranslational modifications of the Ia-associated invariant protein p41 after gene transfer.
Biochemical analysis of a rat fibroblast cell clone, transfected with the murine Ia associated invariant chain gene, demonstrates the expression of a family of proteins. This indicates that all members of the invariant protein family are derived from the same gene. The proteins p41, Ii, p27, p25, and p10, synthesized in the transfectant cell line, are identical with the invariant proteins previously shown to associate noncovalently with Ia antigens. These proteins are identified by immunoprecipitation and western blotting with a monoclonal antibody against the N-terminus and with an antiserum against the C-terminal part of the invariant chain. One protein, p41, has recently been shown to contain a thyroglobulin repeat (TgR) element. It was suggested that p41 might use the TgR element as a signal sequence which guides its intracellular transport to endosomes or lysosomes. Here, I demonstrate that p41 binds four N-linked carbohydrates and is heavily sialylated. During transport through trans Golgi compartments p41 binds palmitic acid, presumably at the same cytoplasmic cysteine as previously shown for Ii. A consensus sequence surrounding the palmitylated cysteine of the invariant chains (Ii, p41) and the human transferrin receptor was found. The transferrin receptor is known to follow an endocytic pathway, for which its cytoplasmic domain is essential. It is conceivable that the palmitylated domain of the invariant chains is a guiding structure for the membrane fusion process with transport vesicles. A role of the invariant proteins for antigen processing/presentation is discussed.
Animals↗
Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo.
Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Human very low density lipoprotein apolipoprotein E isoprotein polymorphism is explained by genetic variation and posttranslational modification.
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Adult↗
Hypoxanthine-guanine phosphoribosyltransferase in human erythroid cells: posttranslational modification.
Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (HGPRT) of human red blood cells has been separated into three major isoenzymes, the relative quantities of which change as the cell ages. The predominant isoenzyme in the youngest circulating red blood cells, reticulocytes, has the same isoelectric point as the single enzyme of lymphoblasts. This lymphoblast-like enzyme is diminished in older red cells, and the major fraction of HGPRT activity is recovered in the two more acidic isoenzymes. The HGPRT enzymes of human lymphoblasts and red cells have been purified to apparent homogeneity, as evidenced by the criterion of subunit molecular weight in NaDodSO4 gels. The lymphoblast enzyme dissociates to a single subunit (alpha) upon isoelectric focusing in 8 M urea and is presumed to be a homo dimer (alpha alpha). The red cell isoenzymes dissociate to two subunits, one with the same isoelectric point as that in lymphoblasts (alpha) and one more negatively charged (alpha'). We infer that the three major red cell isoenzymes, I-III, correspond to enzyme species with none (alpha alpha), one (alpha alpha'), or both (alpha' alpha') subunits modified. Tryptic peptide maps of these iodo[2-14C]acetamide-labeled enzyme subunits indicate that the one red cell subunit (alpha) is identical with that in lymphoblasts and that the second subunit (alpha') differs from these in only one of the five cysteine-containing tryptic peptides. These results indicate that the HGPRT subunit is subject to at least one covalent and site-specific modification in human erythroid cells.
Erythrocyte Aging↗
Tandem mass spectrometry for the examination of the posttranslational modifications of high-mobility group A1 proteins: symmetric and asymmetric dimethylation of Arg25 in HMGA1a protein.
High-mobility group (HMG) A1a and A1b proteins are among a family of HMGA proteins that bind to the minor groove of AT-rich regions of DNA. Here we employed tandem mass spectrometry and determined without ambiguity the sites of phosphorylation and the nature of methylation of HMGA1 proteins that were isolated from the PC-3 human prostate cancer cells. We showed by LC-MS/MS that Ser101 and Ser102 were completely phosphorylated in HMGA1a protein, whereas only a portion of the protein was phosphorylated at Ser98. We also found that the HMGA1b protein was phosphorylated at the corresponding sites, that is, Ser90, Ser91 and Ser87. In addition, Arg25, which is within the first DNA-binding AT-hook domain of HMGA1a, was both mono- and dimethylated. Moreover, both symmetric and asymmetric dimethylations were observed. The closely related HMGA1b protein, however, was not methylated. The unambiguous identification of the sites of phosphorylation and the nature of methylation facilitates the future examination of the biological implications of the HMGA1 proteins.
Amino Acid Sequence↗
Posttranslational modification of oat phytochrome A: phosphorylation of a specific serine in a multiple serine cluster.
Phytochrome A (phyA) is a photoreceptor of higher plants which mediates a variety of biochemical and physiological processes in response to red/far-red light. By detailed structural analysis of the peptides of the total tryptic digest of oat phyA, we found that the photoreceptor isolated from red light irradiated seedlings contains only one site of phosphate attachment, in the N-terminal Ser-rich region. The N-terminal tryptic phosphopeptide (residues 1-12) contains eight serine residues, any of which may be phosphorylated. Direct fast atom bombardment mass spectrometry (FAB MS/MS) analysis of the phosphorylated peptide as well as of its phosphate-containing fragment (residues 1-9) was not successful due to their hydrophilic nature and instability of the phosphate bond. beta-Elimination of the phosphorylated tryptic peptide in the presence of ethanethiol converted the phosphoserine residue to S-ethylcysteine that is stable under FAB MS/MS. FAB MS/MS analysis of the modified peptide clearly showed that the phosphate group was attached to Ser7. The in vivo phosphorylation site at Ser7 in oat phyA is discussed for its possible regulatory role in phyA function.
Amino Acid Sequence↗
Posttranslational modifications of the lutropin receptor: mass spectrometric analysis.
Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.
Amino Acid Sequence↗
Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing.
The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.
Amino Acid Sequence↗
Mammalian sperm tubulin: an exceptionally large number of variants based on several posttranslational modifications.
Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The alpha- and beta-tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in alpha and 0 to 15 in beta. Corresponding values for side-chain glutamyl residues were 0-6 in alpha and 0-3 in beta. Additional alpha variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in alpha- and beta-tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.
Amino Acid Sequence↗
High-sensitivity mass spectrometry for analysis of posttranslational modifications.
Pulsed fast atom bombardment ionization (pulsed-FAB) mass spectrometry has been developed to improve the sensitivity of tandem mass spectrometry (MS/MS), allowing it to be used for the analysis of very small samples. MS/MS, when used with a magnetic four-sector instrument coupled with the pulsed-FAB system, allows significant enhancement in product ion intensity of over ten-fold in magnitude over conventional FAB. MS/MS was applied to the structural analysis of a unique nuclear protein, designated p28, which was isolated from a histone fraction obtained from starfish testes. The results clearly show that protein p28 is a heterodimer composed of testicular histones H2B and H4 which are cross-linked between Gln9 of H2B and Lys5 of H4.
Amino Acid Sequence↗
Glycosylation of microtubule-associated protein tau: an abnormal posttranslational modification in Alzheimer's disease.
Alzheimer's disease (AD) is characterized by the presence of numerous neurons with neurofibrillary tangles of paired helical filaments (PHFs). The microtubule-associated protein tau in abnormally hyperphosphorylated form is the major protein subunit of the PHF. We now show that PHF tangles isolated from AD brains are glycosylated, whereas no glycan is detected in normal tau. Deglycosylation of PHF tangles by endoglycosidase F/N-glycosidase F converts them into bundles of straight filaments 2.5 +/- 0.5 nm in diameter, similar to those generated by the interaction of normal tau and abnormally hyperphosphorylated tau (AD P-tau). Deglycosylation plus dephosphorylation, but not deglycosylation alone, of AD P-tau and tau from PHF tangles restores their microtubule polymerization activity. Dephosphorylation of deglycosylated PHF tangles results in increased tau release. Thus, although the abnormal phosphorylation might promote aggregation of tau and inhibition of the assembly of microtubules, glycosylation appears to be responsible for the maintenance of the PHF structure.
Alzheimer Disease↗
Posttranslational modification of TEL and TEL/AML1 by SUMO-1 and cell-cycle-dependent assembly into nuclear bodies.
The E-26 transforming specific (ETS)-related gene, TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. TEL is a nuclear phosphoprotein that is widely expressed in all normal tissues. TEL contains a DNA-binding domain at the C terminus and a helix-loop-helix domain (also called a pointed domain) at the N terminus. The pointed domain is necessary for homotypic dimerization and for interaction with the ubiquitin-conjugating enzyme UBC9. Here we show that the interaction with UBC9 leads to modification of TEL by conjugating it to SUMO-1. The SUMO-1-modified TEL localizes to cell-cycle-specific nuclear speckles that we named TEL bodies. We also show that the leukemia-associated fusion protein TEL/AML1 is modified by SUMO-1 and found in the TEL bodies, in a pattern quite different from what we observe and report for AML1. Therefore, SUMO-1 modification of TEL could be a critical signal necessary for normal functioning of the protein. In addition, the modification by SUMO-1 of TEL/AML1 could lead to abnormal localization of the fusion protein, which could have consequences that include contribution to neoplastic transformation.
Animals↗